We demonstrate the use of binary colloidal assemblies as lithographic masks to generate tunable Au patterns on SiO2 substrates with dimensions ranging from micrometers to nanometers. Such patterns can be modified with different chemistries to create patterns with well-defined sites for selective adsorption of proteins, where the pattern size and spacing is adjustable depending on particle choice. In our system, the binary colloidal assemblies contain large and small particles of similar or different material and are self-assembled from dilute dispersions with particle size ratios ranging from 0.10 to 0.50. This allows masks with variable morphology and thus production of chemical patterns of tunable geometry. Finally, the Au or SiO2 regions of the pattern are surface modified with protein resistant oligoethyleneglycol self-assembled molecules, which facilitates site selective adsorption of proteins into the unmodified regions of the pattern. This we show with fluorescently labeled bovine serum albumin.